Hepatitis C virus (HCV) is a major world-wide cause of chronic liver disease and hepatic failure for which effective therapies are needed. Within the positive-strand RNA genome of HCV exists an internal ribosomal entry site (IRES) that mediates viral gene expression. Significant efforts have been directed toward characterization of HCV IRES-mediated translation initiation, partly because the HCV IRES presents an attractive target for antiviral therapy. However, a role for specific c^-acting HCV RNA elements in translation control and the function of trans-acting cellular proteins in this process remains obscure. This proposal examines the molecular mechanism of translation initiation at the HCV IRES and its modulationby the 3' nontranslatedregion, the polypyrimidine tract binding protein, and previously unidentified trans-acting factors. Cis- and trans-acting regulatory factors will be characterized through the utilization of a novel cell- based reporter expression system that takes into account the complex involvement of viral and cellular determinants in translationcontrol at the HCV IRES.